Friday 12/6
Ran Inhibition assay. During the first trial, forgot to add inhibitor and positive orthovanidate control. Second trial, added inhibitor, but still forgot the orthovanidate control.
Trial 1:
Trial 2:
Also attempted to do virtual screening with the control ligands. Did not run.
Wednesday 11/27
Concentrated both elutions to 4.26 mg/ml in concentration tube and ran through FPLC.
Figure above: FPLC result for YopH
Tuesday 11/26
Purified Trial 2, nanodropped, and ran on PAGE gel Color Plus prestained Protein Ladder.
Figure above: SDS-PAGE gel of large scale expression of YopH, flask 1
Figure above: SDS-PAGE gel of large scale expression of YopH, flask 1
Monday 11/25
Sonicated the two samples, spun down and saved supernatant. Purified Trial 1 using a used chromatography tube.
Friday 11/22
The next day, transferred to large tubes and centrifuged to retreive pellets, which were resuspended in sonication buffer and put into conical tubes.
Thursday 11/21
Started Option A expression from an available YapH plate to be used as a surrogate. Grew up 2 separate tubes of colonies in the shaking incubator for 8 hours, then measured the OD600 using a spectrophotometer. It never got up to 0.1. Left overnight for 18 hours in biobricks lab in room temperature incubator. I have now realized that the main reason the OD600 never reached the proper values is because instead of growing one of the tube colonies in large scale growth and adding in aliquots of the second tube to reach the proper OD600, we grew up two huge cultures in 2-2L flasks.
Wednesday 11/20
Mini-prepped last batch.
Tuesday 11/19
Made 1 more batch of tubes. Negative results for second set of colonies.
The colonies in the tubes grew very fitfully and therefore had very low concentrations when midiprepped.
Friday 11/15
Miniprepped second batch of tubes and received negative results from the first batch.
Thursday 11/14
Remade batch of tubes with Kanomycin.
Made a list of negative and positive control ligands for virtual.
Wednesday 11/13
Mini-prepped samples and submitted to sequencing.
Sunday 11/10
Decanted and centrifuged tubes, and then realized that I had FORGOTTEN TO ADD KANOMYCIN.
Saturday 11/9
Plate A and Plate B grew small yellow colonies, but B had more colonies.
Made 8 tubes and a master plate from the two plates (#1-4 from plate A and #5-8 from plate B)
Friday 11/8
Cohesive end generation for PCR inserts and Accepting vector.
Annealing and Transformation
Tube A - 2 ul accepting, 4 ul insert
Tube B- 2 ul accepting, 6 ul insert
Wednesday 11/6
PCR cleanup - 43.0 ng/ul
Tuesday 11/5
Prepped pNIC with BSAI-HF, made 2-40 microliter tubes of prepped plasmid.
Friday 11/1/13
Midi prepped pNIC, but achieved low concentration of 7.9 and 8 ng/microliter, so moved on to using Alyssa's prepped plasmid.
Friday 10/25
Centrifuged and decanted pNIC - made 4 tubes.
Thursday 10/24
Virtual setup. Not yet complete.
Restarted pNIC protocols- overnight culture with 160 mL of LB.
Friday 10/18
PCR cleanup on both plasmid and insert. Messed up plasmid PCR cleanup by decanting during the last step, but PCR squared came out fine with a concentration of 58.7 ng/microliter.
Nanodropped both.
Figure above: Nanodrop of remaining PCR-cleaned up pNIC plasmid
Figure above: Nanodrop of PCR-cleaned up PCR squared
Thursday 10/17
Prepared pNIC accepting vector with restriction enzymes
Tuesday 10/15
Midi prepped pNIC
Average concentration: 39.7 g/Microliter
Figure above: Lab notebook picture of Midiprep results
Friday 10/11
Took out and centrifuged pNIC
Figure 1 above: Overnight incubation of pNIC
Figure 2 above: Centrifuged and decanted pNIC
Thursday 10/10
Tested PCR squared with Fermentas gene ruler, SUCCESS!!
Put pNIC transformation in incubator overnight Figure above: 0.7 agarose gel with 100 bp Fermentas gene ruler and PCR squared samples.
Great work updating your wikispace Julia. The captions and analysis look good, but make sure you upload the images properly I cant seem to find them. Where are you exactly with virtual? Thank you. -Max 10/21/13
Tuesday 10/8
Made PCRsquared at 60 degrees and middle times.
Monday 10/7
Made and ran gel of SPCR at 60 degrees and middle times. Success! But PCR tube was crushed by the lid being too tight.
Displaying jmc5736_secondaryPCR#3.JPG
Figure above: Secondary PCR attempt at 60 degrees and middle times. First band: 100 bp ladder. Second band: Visible SPCR smear.
Wednesday 10/2
Ran gel of SPCR, which failed to show bands.
Displaying jmc5736_secondaryPCR#2.JPG
Figure above: Secondary PCR attempt at 56 degrees. First band: 1kbp ladder.
Tuesday 10/1
Ran PPCR gel- band was formed.
Made and left SPCR (56 degrees)
Displaying jmc5736_primaryPCR#2.JPG
Figure above: Primary PCR attempt at 56 degrees: 1st band: 100 bp ladder. 2nd band: successful sample smear.
Monday 9/30
Remade PPCR mix at 56 degrees and ran overnight.
Saturday 9/28
Made SPCR and ran on gel, which tore but also didn't show any bands.
Finished Pymol Refresher
Displaying jmc5736_secondaryPCR.JPG
Figure above: Secondary PCR attempt. 1st band: 100 bp ladder.
Friday 9/27
Ran PPCr again, smear was present
Displaying imransecondary.JPG
Figure above: Primary PCR attempt. 1st band: 100 bp ladder. 3rd band: PPCR.
Thursday 9/26
Made LB Media and attempted Primary PCR, which didn't show any bands or even the ladder.
Julia, you're missing week 5 and 6 :( update your wikispaces! - Michael T.
Week 3 & 4
Julia - ok, need Primary PCR (for your next Wiki), Crop your gel images in the Imaging Software, Add captions to bottom of Figures, Neeed brief analysis, include an image of ladder. - Dr. B 092713
Monday 9/16/13
Completed enzyme digest protocol, and didn't achieve desired results. Lanes 4 and 5 were very faint and did not have the correct amount of lines, meaning the digest did not go to completion.
Figure 1: pGBR22 Enzyme digest gel run. Lane 1: empty. Lane 2: 1kb DNA ladder. Lane 3: EcoRI digest. Lane 4: PvuII digest. Lane 5: EcoRi and PvuII digest. Lane 6: Undigested pGBR22.
Designed Tail primers for pNIC-Bsa4 Cloning
Final result:
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTT
TGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGT
AGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGACGCGCTGACCACCCTCCCGATCAAAAAGCACACCGCGC
TGCTGAACCGTTTCCCG
GAAACCCGCTTCGTTACCCAACTGGCGAAAAAGCGTGCGTCTTGGATCGTTTTCGGTCAC
TACCTCACTCCAGCACAGTTTGAAGATATGGATTTTTTCACCAATCGTTTCAATGCGATC
CTGGACATGTGGAAAGTTGGCCGTTACGAAGTTGCGCTGATGGACGGTGAACTGACCTCT
GAACACGAAACCATCCTGAAAGCGCTGGAACTCGACTACGCTCGCATCCAGGACGTTCCA
GACCTCACCAAACCGGGCCTGATCGTTCTCGACATGGACTCTACCGCTATCCAGATCGAA
TGCATCGACGAAATTGCGAAGCTGGCGGGTGTTGGCGAGGAAGTGGCCGAAGTTACGGAA
CGTGCGATGCAGGGCGAGCTGGACTTCGAACAGTCTCTGCGTCTGCGTGTTTCTAAACTC
AAAGACGCCCCTGAACAGATCCTGAGCCAGGTTCGTGAAACGCTGCCGCTCATGCCTGAA
CTGCCGGAACTGGTTGCGACCCTGCACGCGTTCGGTTGGAAGGTAGCAATCGCGTCTGGT
GGTTTCACCTACTTTTCTGACTACCTGAAGGAACAACTCAGCCTCGATTACGCGCAGTCT
AACACCCTGGAAATTGTTTCTGGTAAACTGACTGGTCAAGTTCTGGGTGAAGTTGTGTCT
GCTCAGACCAAAGCGGACATCCTGCTGACCCTGGCGCAACAGTACGACGTTGAAATCCAC
AACACCGTTGCGGTGGGTGACGGTGCGAACGACCTGGTTATGATGGCGGCTGCGGGCCTC
GGTGTAGCGTACCATGCGAAACCGAAGGTTGAGGCGAAGGCGCAGACCGCAGTTCGTTTC
GCTGGTCTCGGTGGTGTCGTTTGCATCCTGTCTGCGGCGCTCGTTGCGCAGCAAAAACTC
TCTTGGAAATCTAAACCGTAACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCA
CTCGAGCACCACCACCACCACC
ACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTG
AGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGA
AAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGC
GGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGC
TCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCT
AAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAA
ACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCC
TTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACT
CAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTG
GTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTT
TACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTT
CTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCA
TCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCC
GTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGT
ATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAA
AAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCA
AAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAA
AATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATA
CGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACA
CTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATG
CTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAAT
GCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTG
TAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCT
TCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTAT
ACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCC
GTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTG
TTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA
AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACA
AAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTT
CCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG
TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATC
CTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGA
CGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC
AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGC
GCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACA
GGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGG
TTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA
TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCT
CACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAG
TGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAA
GCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGC
ATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACAC
TCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGA
CGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTC
CGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCG
GTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTC
CAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTT
AAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCAT
GGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGA
ACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGA
CCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCC
ACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGA
CTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCA
GGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATT
CTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGAT
CATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTT
GGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAG
CGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAG
CGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGAC
GATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCAT
CGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACT
GCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGC
GGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGG
GCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGC
TGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATG
AGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGG
ACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAG
TGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCC
AGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGC
CAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCT
GGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAA
TAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGC
AGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCAC
TGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTT
CTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGA
CAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACT
GTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCG
CTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAA
CGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACAT
TCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGC
GCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAG
CAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAG
GAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAA
GCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAG
GCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGG
ATCGAGATCTCGATCCCGCGAAAT
9/20/13
Prepared Oligo mix of V. cholarae DNA. Will start primary and secondary PCR next week.
Week 1 & 2
Julia, ok looks good. paste aa sequence as text only (no code). Say what is in each lane for this PCR. Dr. B 090913
Week 2
9/02/13
Today I worked on 3 labs: Quantifying DNA using Nanodrop, Submitting DNA to DNA Sequencing Facility, and I started the PCR Protocol
Results for Nanodrop:
The results from the sequencing facility:
Forward NNNNNNNNNNNNNNNGCGAANNNNGGCCCGACGTNCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTTGATGGTGATGGTGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCANANGTCCNNNNNTGGTGACAGTGAGCTTTACCGTCTGCTCCCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTANTGTCCATTGACCGTGCCTGACATATAAACCTTGTANGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTANTGCGGCCGCCTGCAGTCNANCATATGGGAGANCTCCCAACGCGTTGGANGCATAGCTTGANTANTCTATAGTGTCACTAAATAGCTTGCNTATCATGGTCATAGCTGNTTCCTGNGTNAAATTGTATCCNCTCACNATTCNCACACATACGAGCCGNANCNTNANTGNAAAGCTGGGNNGCTATNNNNNNNNACTCACATTANNCNTNNNNNNNCTGCCNCTTCAGTNGGAANCTNTNNGCNNCTGCNTATGANNNCANNNNNGGGANNNNGTTGNNTATGGNNCTNNCNNTCNCNNTNNNTNANTNNNNNNNNNGNNNTCGNTNNNNNNNGNNNNNNNNNNNNNNNNNANNNNNNNNCNNANCNNNNANNNNAAANNNNNNNNANNNNANNNNCNNANNNCNTNNNNNTTCNNNGNNNNCCCNNNNNN
Reverse NNNNNNGGGNNNNCNNNGAANNNNNANGNNNTNNGNNNNTNNNNTNNNNNNNNTTTNNNNTNNNNGNTNNGNNNNNNNNTNNNNNNNNNNNNNNNNNCNNNNNNNANCGANNNCNNNNNNNNNANTNANNNANNGNGANNGNNAGNNCCATANNCAACNNNNTCCCNNNNNTGNNNTCATANGCAGNNGCNNANAGNTTCCNACTGAAGNGGCAGNNNNNNNANGNNTAATGTGAGTNNNNNNNNATAGCNNCCCAGCTTTNCANTNANGNTNCGGCTCGTATGTGTGNGAATNGTGAGNGGATACAATTTNACNCAGGAANCAGCTATGACCATGATANGCAAGCTATTTAGTGACACTATAGANTANTCAAGCTATGCNTCCAACGCGTTGGGAGNTCTCCCATATGNTNGACTGCAGGCGGCCGCANTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACNTACAAGGTTTATATGTCAGGCACGGTCAATGGACANTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCANNNNNGGACNTNTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCAACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGNACGTCGGGCCNNNNTTCGCNNNNNNNNNNNNNNN
Sequencing results BLASTED against Human:
Sequencing results BLASTED against All:
Results for PCR Protocol:
The pGBR22 gene I sent to sequencing came back with 1,214 base pairs, many of which were labeled N. Taking into account that the PCR process often strips the ends of the DNA, my visible bands were within the ballpark of the expected number of base pairs. (PCR doesn't strip them but rather can start inboard of the start for DNA Sequencing - Dr. B)
Ran Inhibition assay. During the first trial, forgot to add inhibitor and positive orthovanidate control. Second trial, added inhibitor, but still forgot the orthovanidate control.
Trial 1:
Trial 2:
Also attempted to do virtual screening with the control ligands. Did not run.
Wednesday 11/27
Concentrated both elutions to 4.26 mg/ml in concentration tube and ran through FPLC.
Figure above: FPLC result for YopH
Tuesday 11/26
Purified Trial 2, nanodropped, and ran on PAGE gel Color Plus prestained Protein Ladder.
Figure above: SDS-PAGE gel of large scale expression of YopH, flask 1
Figure above: SDS-PAGE gel of large scale expression of YopH, flask 1
Monday 11/25
Sonicated the two samples, spun down and saved supernatant. Purified Trial 1 using a used chromatography tube.
Friday 11/22
The next day, transferred to large tubes and centrifuged to retreive pellets, which were resuspended in sonication buffer and put into conical tubes.
Thursday 11/21
Started Option A expression from an available YapH plate to be used as a surrogate. Grew up 2 separate tubes of colonies in the shaking incubator for 8 hours, then measured the OD600 using a spectrophotometer. It never got up to 0.1. Left overnight for 18 hours in biobricks lab in room temperature incubator. I have now realized that the main reason the OD600 never reached the proper values is because instead of growing one of the tube colonies in large scale growth and adding in aliquots of the second tube to reach the proper OD600, we grew up two huge cultures in 2-2L flasks.
Wednesday 11/20
Mini-prepped last batch.
Tuesday 11/19
Made 1 more batch of tubes. Negative results for second set of colonies.
The colonies in the tubes grew very fitfully and therefore had very low concentrations when midiprepped.
Friday 11/15
Miniprepped second batch of tubes and received negative results from the first batch.
Thursday 11/14
Remade batch of tubes with Kanomycin.
Made a list of negative and positive control ligands for virtual.
Wednesday 11/13
Mini-prepped samples and submitted to sequencing.
Sunday 11/10
Decanted and centrifuged tubes, and then realized that I had FORGOTTEN TO ADD KANOMYCIN.
Saturday 11/9
Plate A and Plate B grew small yellow colonies, but B had more colonies.
Made 8 tubes and a master plate from the two plates (#1-4 from plate A and #5-8 from plate B)
Friday 11/8
Cohesive end generation for PCR inserts and Accepting vector.
Annealing and Transformation
Tube A - 2 ul accepting, 4 ul insert
Tube B- 2 ul accepting, 6 ul insert
Wednesday 11/6
PCR cleanup - 43.0 ng/ul
Tuesday 11/5
Prepped pNIC with BSAI-HF, made 2-40 microliter tubes of prepped plasmid.
Friday 11/1/13
Midi prepped pNIC, but achieved low concentration of 7.9 and 8 ng/microliter, so moved on to using Alyssa's prepped plasmid.
Friday 10/25
Centrifuged and decanted pNIC - made 4 tubes.
Thursday 10/24
Virtual setup. Not yet complete.
Restarted pNIC protocols- overnight culture with 160 mL of LB.
Friday 10/18
PCR cleanup on both plasmid and insert. Messed up plasmid PCR cleanup by decanting during the last step, but PCR squared came out fine with a concentration of 58.7 ng/microliter.
Nanodropped both.
Figure above: Nanodrop of remaining PCR-cleaned up pNIC plasmid
Figure above: Nanodrop of PCR-cleaned up PCR squared
Thursday 10/17
Prepared pNIC accepting vector with restriction enzymes
Tuesday 10/15
Midi prepped pNIC
Average concentration: 39.7 g/Microliter
Figure above: Lab notebook picture of Midiprep results
Friday 10/11
Took out and centrifuged pNIC
Figure 1 above: Overnight incubation of pNIC
Figure 2 above: Centrifuged and decanted pNIC
Thursday 10/10
Tested PCR squared with Fermentas gene ruler, SUCCESS!!
Put pNIC transformation in incubator overnight
Figure above: 0.7 agarose gel with 100 bp Fermentas gene ruler and PCR squared samples.
Great work updating your wikispace Julia. The captions and analysis look good, but make sure you upload the images properly I cant seem to find them. Where are you exactly with virtual? Thank you. -Max 10/21/13
Tuesday 10/8
Made PCRsquared at 60 degrees and middle times.
Monday 10/7
Made and ran gel of SPCR at 60 degrees and middle times. Success! But PCR tube was crushed by the lid being too tight.
Figure above: Secondary PCR attempt at 60 degrees and middle times. First band: 100 bp ladder. Second band: Visible SPCR smear.
Wednesday 10/2
Ran gel of SPCR, which failed to show bands.
Figure above: Secondary PCR attempt at 56 degrees. First band: 1kbp ladder.
Tuesday 10/1
Ran PPCR gel- band was formed.
Made and left SPCR (56 degrees)
Figure above: Primary PCR attempt at 56 degrees: 1st band: 100 bp ladder. 2nd band: successful sample smear.
Monday 9/30
Remade PPCR mix at 56 degrees and ran overnight.
Saturday 9/28
Made SPCR and ran on gel, which tore but also didn't show any bands.
Finished Pymol Refresher
Figure above: Secondary PCR attempt. 1st band: 100 bp ladder.
Friday 9/27
Ran PPCr again, smear was present
Figure above: Primary PCR attempt. 1st band: 100 bp ladder. 3rd band: PPCR.
Thursday 9/26
Made LB Media and attempted Primary PCR, which didn't show any bands or even the ladder.
Julia, you're missing week 5 and 6 :( update your wikispaces! - Michael T.
Week 3 & 4
Julia - ok, need Primary PCR (for your next Wiki), Crop your gel images in the Imaging Software, Add captions to bottom of Figures, Neeed brief analysis, include an image of ladder. - Dr. B 092713
Monday 9/16/13
Completed enzyme digest protocol, and didn't achieve desired results. Lanes 4 and 5 were very faint and did not have the correct amount of lines, meaning the digest did not go to completion.
Figure 1: pGBR22 Enzyme digest gel run. Lane 1: empty. Lane 2: 1kb DNA ladder. Lane 3: EcoRI digest. Lane 4: PvuII digest. Lane 5: EcoRi and PvuII digest. Lane 6: Undigested pGBR22.
Designed Tail primers for pNIC-Bsa4 Cloning
Final result:
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTT
TGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGT
AGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGACGCGCTGACCACCCTCCCGATCAAAAAGCACACCGCGC
TGCTGAACCGTTTCCCG
GAAACCCGCTTCGTTACCCAACTGGCGAAAAAGCGTGCGTCTTGGATCGTTTTCGGTCAC
TACCTCACTCCAGCACAGTTTGAAGATATGGATTTTTTCACCAATCGTTTCAATGCGATC
CTGGACATGTGGAAAGTTGGCCGTTACGAAGTTGCGCTGATGGACGGTGAACTGACCTCT
GAACACGAAACCATCCTGAAAGCGCTGGAACTCGACTACGCTCGCATCCAGGACGTTCCA
GACCTCACCAAACCGGGCCTGATCGTTCTCGACATGGACTCTACCGCTATCCAGATCGAA
TGCATCGACGAAATTGCGAAGCTGGCGGGTGTTGGCGAGGAAGTGGCCGAAGTTACGGAA
CGTGCGATGCAGGGCGAGCTGGACTTCGAACAGTCTCTGCGTCTGCGTGTTTCTAAACTC
AAAGACGCCCCTGAACAGATCCTGAGCCAGGTTCGTGAAACGCTGCCGCTCATGCCTGAA
CTGCCGGAACTGGTTGCGACCCTGCACGCGTTCGGTTGGAAGGTAGCAATCGCGTCTGGT
GGTTTCACCTACTTTTCTGACTACCTGAAGGAACAACTCAGCCTCGATTACGCGCAGTCT
AACACCCTGGAAATTGTTTCTGGTAAACTGACTGGTCAAGTTCTGGGTGAAGTTGTGTCT
GCTCAGACCAAAGCGGACATCCTGCTGACCCTGGCGCAACAGTACGACGTTGAAATCCAC
AACACCGTTGCGGTGGGTGACGGTGCGAACGACCTGGTTATGATGGCGGCTGCGGGCCTC
GGTGTAGCGTACCATGCGAAACCGAAGGTTGAGGCGAAGGCGCAGACCGCAGTTCGTTTC
GCTGGTCTCGGTGGTGTCGTTTGCATCCTGTCTGCGGCGCTCGTTGCGCAGCAAAAACTC
TCTTGGAAATCTAAACCGTAACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCA
CTCGAGCACCACCACCACCACC
ACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTG
AGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGA
AAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGC
GGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGC
TCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCT
AAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAA
ACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCC
TTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACT
CAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTG
GTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTT
TACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTT
CTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCA
TCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCC
GTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGT
ATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAA
AAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCA
AAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAA
AATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATA
CGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACA
CTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATG
CTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAAT
GCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTG
TAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCT
TCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTAT
ACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCC
GTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTG
TTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA
AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACA
AAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTT
CCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG
TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATC
CTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGA
CGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC
AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGC
GCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACA
GGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGG
TTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA
TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCT
CACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAG
TGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAA
GCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGC
ATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACAC
TCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGA
CGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTC
CGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCG
GTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTC
CAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTT
AAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCAT
GGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGA
ACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGA
CCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCC
ACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGA
CTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCA
GGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATT
CTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGAT
CATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTT
GGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAG
CGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAG
CGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGAC
GATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCAT
CGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACT
GCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGC
GGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGG
GCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGC
TGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATG
AGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGG
ACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAG
TGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCC
AGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGC
CAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCT
GGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAA
TAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGC
AGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCAC
TGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTT
CTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGA
CAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACT
GTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCG
CTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAA
CGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACAT
TCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGC
GCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAG
CAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAG
GAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAA
GCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAG
GCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGG
ATCGAGATCTCGATCCCGCGAAAT
9/20/13
Prepared Oligo mix of V. cholarae DNA. Will start primary and secondary PCR next week.
Week 1 & 2
Julia, ok looks good. paste aa sequence as text only (no code). Say what is in each lane for this PCR. Dr. B 090913
8/30/13
Today I was assigned my target:
phosphoserine phosphatase (Vibrio cholerae)
Gene: YP_005334106.1
The aa sequence was found on NCBI, so it was different than the original information page on wikispaces.
1 <span class="ff_line">mdalttlpik khtallnrfp etrfvtqlak kraswivfgh yltpaqfedm dfftnrfnai</span> 61 <span class="ff_line">ldmwkvgrye valmdgelts ehetilkale ldyariqdvp dltkpglivl dmdstaiqie</span> 121 <span class="ff_line">cideiaklag vgeevaevte ramqgeldfe qslrlrvskl kdapeqilsq vretlplmpe</span> 181 <span class="ff_line">lpelvatlha fgwkvaiasg gftyfsdylk eqlsldyaqs ntleivsgkl tgqvlgevvs</span> 241 <span class="ff_line">aqtkadillt laqqydveih ntvavgdgan dlvmmaaagl gvayhakpkv eakaqtavrf</span> 301 <span class="ff_line">aglggvvcil saalvaqqkl swkskp</span>I ordered the oligos under the order VcPP_1. The modified sequence was:1 ATGGACGCGCTGACCAC 17
2 CCGGGAAACGGTTCAGGAGCGCGGTGTGTTTTTTGATCGGCAGCGTGGTCAGCGCGTCCA 60
3 TCCTGAACCGTTTCCCGGAAACCCGTTTCGTTACGCAACTGGCGAAGAAACGCGCGAGCT 60
4 TCTTCAAACTGTGCCGGGGTCAGATAGTGACCGAAAACGATCCAGCTCGCGCGTTTCTTC 60
5 CCCGGCACAGTTTGAAGATATGGACTTCTTCACCAATCGCTTTAATGCCATCCTCGATAT 60
6 CGTCCATGAGCGCAACCTCATAACGACCCACTTTCCACATATCGAGGATGGCATTAAAGC 60
7 GGTTGCGCTCATGGACGGTGAACTCACCTCTGAACACGAAACCATTCTGAAGGCGCTGGA 60
8 TTGGTGAGGTCCGGAACGTCCTGGATACGTGCGTAATCGAGTTCCAGCGCCTTCAGAATG 60
9 CGTTCCGGACCTCACCAAACCGGGTCTCATCGTTCTGGACATGGATTCTACCGCGATTCA 60
10 ACACCCGCCAGCTTCGCGATTTCGTCGATGCATTCGATCTGAATCGCGGTAGAATCCATG 60
11 CGAAGCTGGCGGGTGTCGGTGAGGAAGTTGCGGAAGTTACCGAACGTGCTATGCAGGGCG 60
12 CAGTTTAGAAACACGGAGACGCAGAGACTGTTCGAAATCCAGTTCGCCCTGCATAGCACG 60
13 CGTCTCCGTGTTTCTAAACTGAAGGATGCACCGGAACAGATCCTGAGCCAAGTTCGTGAA 60
14 GTCGCAACGAGCTCTGGCAGTTCCGGCATCAGCGGCAGGGTTTCACGAACTTGGCTCAGG 60
15 CCAGAGCTCGTTGCGACCCTGCACGCATTCGGTTGGAAGGTAGCAATCGCCTCCGGTGGT 60
16 CCAGAGAGAGCTGCTCTTTCAGGTAGTCGCTGAAGTAGGTAAAACCACCGGAGGCGATTG 60
17 GAAAGAGCAGCTCTCTCTGGACTATGCGCAGTCTAACACCCTCGAAATTGTTTCTGGTAA 60
18 GCGCGGAGACAACTTCACCGAGAACCTGACCAGTGAGTTTACCAGAAACAATTTCGAGGG 60
19 GTGAAGTTGTCTCCGCGCAGACCAAAGCGGACATCCTGCTGACGCTCGCCCAGCAGTATG 60
20 TTCGCGCCATCGCCAACCGCAACGGTGTTGTGAATTTCAACGTCATACTGCTGGGCGAGC 60
21 TGGCGATGGCGCGAACGACCTGGTTATGATGGCGGCTGCGGGCCTGGGTGTGGCCTACCA 60
22 ACGAACCGCCGTTTGCGCCTTTGCTTCGACTTTCGGTTTCGCGTGGTAGGCCACACCCAG 60
23 GCAAACGGCGGTTCGTTTTGCGGGTCTGGGTGGTGTCGTTTGTATTCTGTCTGCCGCGCT 60
24 TTACGGTTTAGATTTCCAAGACAGTTTTTGCTGCGCCACCAGCGCGGCAGACAGAAT 57
Week 2
9/02/13
Today I worked on 3 labs: Quantifying DNA using Nanodrop, Submitting DNA to DNA Sequencing Facility, and I started the PCR Protocol
Results for Nanodrop:
The results from the sequencing facility:
Forward
NNNNNNNNNNNNNNNGCGAANNNNGGCCCGACGTNCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTTGATGGTGATGGTGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCANANGTCCNNNNNTGGTGACAGTGAGCTTTACCGTCTGCTCCCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTANTGTCCATTGACCGTGCCTGACATATAAACCTTGTANGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTANTGCGGCCGCCTGCAGTCNANCATATGGGAGANCTCCCAACGCGTTGGANGCATAGCTTGANTANTCTATAGTGTCACTAAATAGCTTGCNTATCATGGTCATAGCTGNTTCCTGNGTNAAATTGTATCCNCTCACNATTCNCACACATACGAGCCGNANCNTNANTGNAAAGCTGGGNNGCTATNNNNNNNNACTCACATTANNCNTNNNNNNNCTGCCNCTTCAGTNGGAANCTNTNNGCNNCTGCNTATGANNNCANNNNNGGGANNNNGTTGNNTATGGNNCTNNCNNTCNCNNTNNNTNANTNNNNNNNNNGNNNTCGNTNNNNNNNGNNNNNNNNNNNNNNNNNANNNNNNNNCNNANCNNNNANNNNAAANNNNNNNNANNNNANNNNCNNANNNCNTNNNNNTTCNNNGNNNNCCCNNNNNN
Reverse
NNNNNNGGGNNNNCNNNGAANNNNNANGNNNTNNGNNNNTNNNNTNNNNNNNNTTTNNNNTNNNNGNTNNGNNNNNNNNTNNNNNNNNNNNNNNNNNCNNNNNNNANCGANNNCNNNNNNNNNANTNANNNANNGNGANNGNNAGNNCCATANNCAACNNNNTCCCNNNNNTGNNNTCATANGCAGNNGCNNANAGNTTCCNACTGAAGNGGCAGNNNNNNNANGNNTAATGTGAGTNNNNNNNNATAGCNNCCCAGCTTTNCANTNANGNTNCGGCTCGTATGTGTGNGAATNGTGAGNGGATACAATTTNACNCAGGAANCAGCTATGACCATGATANGCAAGCTATTTAGTGACACTATAGANTANTCAAGCTATGCNTCCAACGCGTTGGGAGNTCTCCCATATGNTNGACTGCAGGCGGCCGCANTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACNTACAAGGTTTATATGTCAGGCACGGTCAATGGACANTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCANNNNNGGACNTNTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCAACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGNACGTCGGGCCNNNNTTCGCNNNNNNNNNNNNNNN
Sequencing results BLASTED against Human:
Sequencing results BLASTED against All:
Results for PCR Protocol:
The pGBR22 gene I sent to sequencing came back with 1,214 base pairs, many of which were labeled N. Taking into account that the PCR process often strips the ends of the DNA, my visible bands were within the ballpark of the expected number of base pairs. (PCR doesn't strip them but rather can start inboard of the start for DNA Sequencing - Dr. B)